Anti-vla-4 related assays

ABSTRACT

Methods and apparatus for assaying the level of analytes in a sample, related to VLA-4, are disclosed. A method of decreasing the level of an anti-integrin antibody in a subject is described including a) contacting a biological sample from a subject with a detectable capture agent associated with a substrate, wherein the capture agent can bind an anti-integrin antibody in the sample; b) detecting binding of the capture agent with the level of the anti-integrin antibody; and c) treating the subject with plasma exchange until the level of the anti-integrin antibody in the sample reaches a predetermined level.

CLAIM OF PRIORITY

This application claims priority to provisional U.S. Patent ApplicationNo. 61/250,553, filed on Oct. 11, 2009, which is incorporated byreference in its entirety.

FIELD OF THE INVENTION

The invention relates to assays associated with immuno-modulatorytherapies SUMMARY OF THE INVENTION

The invention is based, at least in part, on the development of a testto assay the level of an anti-integrin antibody and theanti-(anti-integrin antibody) antibody, e.g., anti-drug antibody, in asample.

In one aspect, a method of decreasing the level of an anti-integrinantibody in a subject includes contacting a biological sample from asubject with a detectable capture agent associated with a substrate,wherein the capture agent can bind an anti-integrin antibody in thesample, detecting binding of the capture agent with a level of theanti-integrin antibody in the sample, and treating the subject withplasma exchange until the level of the anti-integrin antibody in thesample reaches a predetermined level. The method can include a subjectwho was treated with a natalizumab and has been diagnosed with or issuspected of having a JC virus infection.

Certain embodiments can include one or more of the following features.Contacting the biological sample with a detectable capture agent anddetecting binding of the capture agent can include assaying in a lateralflow system. The binding of the capture agent with the antibody in thesample can produce a detectable signal. Binding of the capture agentwith the antibody can be detected by a secondary agent. Thepredetermined level of the antibody can be about 1 μg/ml, 2 μg/ml, 3μg/ml, 4 μg/ml, 5 μg/ml, 6 μg/ml, 7 μg/ml, 8 μg/ml, 9 μg/ml, 10 μg/ml,15 μg/ml, or 20 μg/ml. Contacting a biological sample from a subjectwith a detectable capture agent associated with a substrate, anddetecting binding of the capture agent to the antibody can occur oneday, two days, three days, four days, five days, six days, one week, twoweeks, three weeks, or four weeks after the plasma exchange treatment.The plasma exchange treatment can be repeated once a day, or once everytwo days, three days, four days, five days, six days, one week, twoweeks, three weeks, or four weeks. The biological sample can be blood,plasma, serum, urine, saliva, cerebrospinal fluid, sputum, ocular lensfluid, sweat, milk, ascites fluid, mucous, synovial fluid, peritonealfluid, transdermal exudates, pharyngeal exudates, bronchoalveolarlavage, tracheal aspirations, semen, cervical mucus, vaginal secretion,urethral secretion, or amniotic fluid. The anti-integrin antibody can bea recombinant anti-integrin antibody or an anti-alpha4 integrin chainantibody. The capture agent can be an antibody or an integrin.

In another aspect, an apparatus for assaying the level of ananti-integrin antibody in a biological sample includes a substrate, anda detectable capture agent associated with the substrate, wherein thecapture agent can bind an anti-integrin antibody in the biologicalsample.

Certain embodiments can include one or more of the following features.The apparatus can include a lateral flow system. The binding of thecapture agent to the anti-integrin antibody can produce a detectablesignal. The binding of the capture agent to the anti-integrin antibodycan be detected by a secondary agent. The capture agent can be anantibody or an integrin. The anti-integrin antibody can be a recombinantanti-integrin antibody. The recombinant anti-integrin antibody is ananti-alpha4 integrin chain antibody. The substrate can benitrocellulose, cellulose acetate, filter paper, cloth, or glass fiberpaper. The biological sample can be blood, plasma, serum, urine, saliva,cerebrospinal fluid, sputum, ocular lens fluid, sweat, milk, ascitesfluid, mucous, synovial fluid, peritoneal fluid, transdermal exudates,pharyngeal exudates, bronchoalveolar lavage, tracheal aspirations,semen, cervical mucus, vaginal secretion, urethral secretion, oramniotic fluid. The binding of the capture agent to the antibody can bedetectable within about 30 seconds to about 60 minutes or longer afterthe binding.

In another aspect, a method of assaying the level of an anti-integrinantibody in a biological sample includes obtaining a biological samplefrom a subject, contacting the sample with a detectable capture agentassociated with a substrate, wherein the capture agent can bind ananti-integrin antibody in the sample, and detecting and correlatingbinding of the capture agent to the level of the anti-integrin antibody.The method can include a subject who was treated with a natalizumab andhas been diagnosed with or is suspected of having a JC virus infection.

Certain embodiments can include one or more of the following features.Contacting the sample with a detectable capture agent and detecting thebinding of the capture agent can include assaying in a lateral flowsystem. The binding of the capture agent to the antibody can produce adetectable signal. Binding of the capture agent to the antibody can bedetected by a secondary agent. The method can include administering atreatment to the subject to decrease the level of the anti-integrinantibody in the subject. The treatment can be plasma exchange. The levelof the anti-integrin antibody can be measured one day, two days, threedays, four days, five days, six days, one week, two weeks, three weeks,or four weeks after the administration of the treatment. Administeringthe treatment and measuring the level of the anti-integrin antibody canbe repeated until the level of the anti-integrin antibody in the samplereaches a predetermined level. The predetermined level can be about 1μg/ml, 2 μg/ml, 3 μg/ml, 4 g/ml, 5 μg/ml, 6 μg/ml, 7 μg/ml, 8 μg/ml, 9μg/ml, 10 μg/ml, 15 μg/ml, or 20 μg/ml. The treatments can beadministered once a day, or once every two days, three days, four days,five days, six days, one week, two weeks, three weeks, or four weeks.The anti-integrin antibody can be a recombinant anti-integrin antibody.The anti-integrin antibody can be an anti-alpha4 integrin chainantibody. The capture agent can be an antibody or an integrin. Thebiological sample can be blood, plasma, serum, urine, saliva,cerebrospinal fluid, sputum, ocular lens fluid, sweat, milk, ascitesfluid, mucous, synovial fluid, peritoneal fluid, transdermal exudates,pharyngeal exudates, bronchoalveolar lavage, tracheal aspirations,semen, cervical mucus, vaginal secretion, urethral secretion, oramniotic fluid.

In another aspect, a method of assaying the efficacy of a treatment canbe obtaining a biological sample from a subject undergoing treatment todecrease the level of an anti-integrin antibody in the subject,contacting the sample with a detectable capture agent associated with asubstrate, wherein the capture agent can bind an anti-integrin antibodyin the sample, and detecting and correlating the binding of the captureagent to the level of the anti-integrin antibody, wherein a level of theanti-integrin antibody in the sample less than a predetermined level isindicative of the efficacy of the treatment. The method can include asubject who was treated with a natalizumab and has been diagnosed withor is suspected of having a JC virus infection.

Certain embodiments can include one or more of the following features.Contacting the sample with a detectable capture agent and detecting andcorrelating the binding of the capture agent can include assaying in alateral flow system. The binding of the capture agent to the antibodycan produce a detectable signal. Binding of the capture agent to theantibody can be detected by a secondary agent. The predetermined levelcan be about 1 μg/ml, 2 μg/ml, 3 μg/ml, 4 μg/ml, 5 μg/ml, 6 μg/ml, 7μg/ml, 8 μg/ml, 9 μg/ml, 10 μg/ml, 15 μg/ml, or 20 μg/ml. The biologicalsample can be obtained from the subject one day, two days, three days,four days, five days, six days, one week, two weeks, three weeks, orfour weeks after the treatment. The treatment can be repeated once aday, or once every two days, three days, four days, five days, six days,one week, two weeks, three weeks, or four weeks. The treatment caninclude plasma exchange. The biological sample can be blood, plasma,serum, urine, saliva, cerebrospinal fluid, sputum, ocular lens fluid,sweat, milk, ascites fluid, mucous, synovial fluid, peritoneal fluid,transdermal exudates, pharyngeal exudates, bronchoalveolar lavage,tracheal aspirations, semen, cervical mucus, vaginal secretion, urethralsecretion, or amniotic fluid. The anti-integrin antibody can be arecombinant anti-integrin antibody. The anti-integrin antibody can be ananti-alpha4 antibody. The capture agent can be an antibody or anintegrin.

In another aspect, a kit for assaying the level of an anti-integrinantibody in a biological sample for use in treating with measuring thelevel of an anti-integrin antibody in a subject and monitoring theefficacy of the treatment includes a tester that includes a substrateand a detectable capture agent associated with the substrate. Thecapture agent can bind an anti-integrin antibody in the biologicalsample and a chase buffer. The subject can include a subject who wastreated with a natalizumab and has been diagnosed with or is suspectedof having a JC virus infection. The kit can further include instructionsfor interpreting the level of the anti-integrin antibody. The kit canalso be used to monitor the efficacy of treatment in the subject.

Certain embodiments of the kit can include one or more of the followingfeatures. The substrate can be a portion of a lateral flow system. Thebinding of the capture agent to the antibody can produce a detectablesignal. The binding of the capture agent to the antibody can be detectedby a secondary agent. The capture agent can be an antibody or anintegrin. The anti-integrin antibody can be a recombinant anti-integrinantibody. The recombinant anti-integrin antibody can be an anti-alpha4antibody. The substrate can be nitrocellulose, cellulose acetate, filterpaper, cloth, or glass fiber paper. The biological sample can be blood,plasma, serum, urine, saliva, cerebrospinal fluid, sputum, ocular lensfluid, sweat, milk, ascites fluid, mucous, synovial fluid, peritonealfluid, transdermal exudates, pharyngeal exudates, bronchoalveolarlavage, tracheal aspirations, semen, cervical mucus, vaginal secretion,urethral secretion, or amniotic fluid. The binding of the capture agentto the antibody can be detectable within about 30 seconds to about 60minutes or longer after the binding.

In another aspect, a method of assaying the efficacy of a treatment canbe obtaining a biological sample from a subject undergoing treatmentwith anti-VLA4 antibody, contacting the sample with a detectable captureagent associated with a substrate, wherein the capture agent can bind ananti-anti-integrin antibody in the sample, and detecting and correlatingthe binding of the capture agent to the level of the anti-anti-integrinantibody to determine the efficacy of the treatment.

Certain embodiments can include one or more of the following features.Contacting the sample with a detectable capture agent and detecting andcorrelating the binding of the capture agent can include assaying in alateral flow system. The binding of the capture agent to the antibodycan produce a detectable signal. Binding of the capture agent to theantibody can be detected by a secondary agent. The biological sample canbe obtained from the subject prior to treatment or one day, two days,three days, four days, five days, six days, one week, two weeks, threeweeks, or four weeks after the treatment. Obtaining the biologicalsample can be repeated once a day, or once every two days, three days,four days, five days, six days, one week, two weeks, three weeks, orfour weeks. The biological sample can be blood, plasma, serum, urine,saliva, cerebrospinal fluid, sputum, ocular lens fluid, sweat, milk,ascites fluid, mucous, synovial fluid, peritoneal fluid, transdermalexudates, pharyngeal exudates, bronchoalveolar lavage, trachealaspirations, semen, cervical mucus, vaginal secretion, urethralsecretion, or amniotic fluid. The anti-anti-integrin antibody can be arecombinant anti-anti-integrin antibody. The anti-anti-integrin antibodycan be an anti-anti-alpha4 integrin chain antibody. The capture agentcan be an antibody.

In another aspect, a kit for assaying the level of an anti-anti-VLA4antibody in a biological sample and monitoring the efficacy of anti-VLA4antibody treatment in a subject includes a tester that includes asubstrate and a detectable capture agent associated with the substrate,wherein the capture agent can bind an anti-anti-VLA4 antibody in thebiological sample and a chase buffer. The kit can further includeinstructions for interpreting the level of the anti-anti-VLA4 antibody.The kit can also be used to monitor the efficacy of treatment in thesubject.

In certain embodiments the kit can include one or more of the followingfeatures. The substrate can be a portion of a lateral flow system. Thebinding of the capture agent to the antibody can produce a detectablesignal. The binding of the capture agent to the antibody can be detectedby a secondary agent. The capture agent can be an antibody. Thesubstrate can be nitrocellulose, cellulose acetate, filter paper, cloth,or glass fiber paper. The biological sample can be blood, plasma, serum,urine, saliva, cerebrospinal fluid, sputum, ocular lens fluid, sweat,milk, ascites fluid, mucous, synovial fluid, peritoneal fluid,transdermal exudates, pharyngeal exudates, bronchoalveolar lavage,tracheal aspirations, semen, cervical mucus, vaginal secretion, urethralsecretion, or amniotic fluid. The binding of the capture agent to theantibody can be detectable within about 30 seconds to about 60 minutesor longer after the binding.

In another aspect, a method of detecting anti-drug antibodies in abiological sample can be obtaining a biological sample from a subjectundergoing treatment with anti-VLA4 antibody, contacting the sample witha detectable capture agent associated with a substrate, wherein thecapture agent can bind an anti-anti-integrin antibody in the sample, anddetecting and correlating the binding of the capture agent to the levelof the anti-anti-integrin antibody to determine the efficacy of thetreatment.

In another aspect, a kit for detecting anti-anti-VLA4 antibody in abiological sample includes a tester that includes a substrate and adetectable capture agent associated with the substrate, wherein thecapture agent can bind an anti-anti-VLA4 antibody in the biologicalsample and a chase buffer.

Definitions

The articles “a” and “an” are used herein to refer to one or to morethan one (i.e., to at least one) of the grammatical object of thearticle. By way of example, “an element” means one element or more thanone element.

The term “about” is used herein to mean a value ±20% of a givennumerical value. Thus, “about 60%” means a value of between 60±(20% of60) (i.e., between 48 and 70).

The term “or” is used herein to mean, and is used interchangeably with,the term “and/or,” unless context clearly indicates otherwise.

As used herein, the term “purify,” “purified,” or “purification” meansthe removal or isolation of a molecule from its environment by, forexample, isolation or separation. “Substantially purified” molecules areat least about 60% free, for example, at least about 75% free, or atleast about 90% free from other components with which they areassociated. In some cases, a substantially purified molecule is at least60% free, 75% free, 90%, or 95% free from other components. As usedherein, these terms also refer to the removal of contaminants from asample.

As used herein, “sample” refers to anything that may contain an analytefor which an analyte assay is desired. The sample may be a biologicalsample, such as a biological fluid or a biological tissue. Examples ofbiological fluids include blood, serum, plasma, saliva, sputum, ocularlens fluid, sweat, urine, milk, ascites fluid, mucous, synovial fluid,peritoneal fluid, transdermal exudates, pharyngeal exudates,bronchoalveolar lavage, tracheal aspirations, cerebrospinal fluid,semen, cervical mucus, vaginal or urethral secretions, amniotic fluid orthe like. Biological tissues are aggregate of cells, usually of aparticular kind together with their intercellular substance that formone of the structural materials of a human, animal, plant, bacterial,fungal or viral structure, including connective, epithelium, muscle andnerve tissues. Examples of biological tissues also include organs,tumors, lymph nodes, arteries and individual cell(s).

As used herein, “fluid sample” refers to a material suspected ofcontaining an analyte(s) of interest, which material has sufficientfluidity to flow through a device or assay described herein. A fluidsample can be obtained from a source and used directly in an assaydescribed herein or can be pretreated so as to modify its character.Such samples can include human, animal or man-made samples. The samplecan be prepared in any medium that does not interfere with an assaydescribed herein. Typically, the sample is an aqueous solution orbiological fluid described herein.

A fluid sample can be derived from any source, such as a physiologicalfluid, including blood, serum, plasma, saliva, sputum, ocular lensfluid, sweat, urine, milk, ascites fluid, mucous, synovial fluid,peritoneal fluid, transdermal exudates, pharyngeal exudates,bronchoalveolar lavage, tracheal aspirations, cerebrospinal fluid,semen, cervical mucus, vaginal or urethral secretions, amniotic fluid,and the like. Fluid homogenates of cellular tissues such as, forexample, hair, skin and nail scrapings. In addition, a solid material tobe assayed for the presence of an analyte can be used as a test sampleonce it is modified to form a liquid medium, e.g., and extract, or torelease the analyte.

As used herein, “antigen” means any compound capable of binding to anantibody, or against which antibodies can be raised.

As used herein, “antibody” refers to a polypeptide substantially encodedby an immunoglobulin gene or immunoglobulin genes, or fragments thereof.Recognized immunoglobulin genes include the kappa, lambda, alpha, gamma,delta, epsilon, and mu constant regions, as well as myriadimmunoglobulin variable region genes. Light chains are classified aseither kappa or lambda. Heavy chains are classified as gamma, mu, alpha,delta, or epsilon, which in turn define the immunoglobulin classes IgG,IgM, IgA, IgD, and IgE, respectively. Typically, an antibody is animmunoglobulin having an area on its surface or in a cavity thatspecifically binds to and is thereby defined as complementary with aparticular spatial and polar organization of another molecule. Theantibody can be polyclonal or monoclonal. Antibodies may include acomplete immunoglobulin or fragments thereof. Fragments thereof caninclude, for example, Fab, Fab′, F(ab′)₂, Fv, single chain Fv (scFv),and the like. Antibodies may also include chimeric antibodies orfragment thereof made by recombinant methods. In some cases the detectedantibody is an IgG4 antibody. In an assay as described herein, IgG4 halfantibodies are detected, complete IgG4 antibodies are detected, or bothIgG4 half antibodies and IgG4 complete antibodies are detected.

As used herein, the term “analyte” refers to a known or unknowncomponent of a sample, which will specifically bind to a capture agentif the analyte and the capture agent are members of a specific bindingpair. In general, analytes are biopolymers, e.g., an oligomer or polymersuch as an oligonucleotide, a polypeptide, (e.g., an antibody), or thelike. In some embodiments, an analyte can exist in a mobile phase (suchas in a fluid) to be detected by a capture agent.

As used herein, the term “labeled reagent” refers to a substancecomprising a detectable label attached to a specific binding agent. Theattachment may be covalent or non-covalent binding, but the method ofattachment is not critical. The label allows the labeled reagent toproduce a detectable signal that is related to the presence of ananalyte in a sample described herein. The specific binding agentcomponent of the labeled reagent is selected to directly or indirectlybind to an analyte. The labeled reagent can be associated with asubstrate described herein, it can be combined with a fluid sample toform a fluid solution, it can be added to a substrate separately from asample, or it can be predeposited or reversibly immobilized on thesubstrate. In addition, the specific binding agent may be labeled beforeor during the performance of an assay described herein by means of anysuitable attachment method known in the art. The detectable label can bea directly detectable label or a label that is detected using indirectmethods.

As used herein, the term “detectable signal” refers to a signal producedby the binding of a capture agent with an analyte. In some embodiments,a detectable signal is detectable by visual inspection. Withoutlimitation, the type of signal produced depends on the label used.Generally, detectable signals indicating the presence or absence of ananalyte in a sample may be evident of their own accord, e.g., detectableas lines, plus or minus signs, or particularly shaped symbols, or may beevident through comparison with a reference, such as a color indicatorreference.

As used herein, the term “capture agent” refers to an agent that bindsan analyte through an interaction that is sufficient to permit the agentto specifically bind an analyte. For example, the capture agent canspecifically bind an analyte with a dissociation constant (K_(d)) ofless than about 10⁻⁶ M without binding to other targets. The bindinginteraction can be mediated by a binding site of the capture agent.Capture agents can include, e.g., any polypeptide, e.g., an antibody.Capture agents can be bound to a substrate or can be present insolution.

As used herein, the term “specific binding” refers to preferentialbinding of an agent to a particular analyte that is present in a mixtureof different analytes. Typically, specific binding differentiatesbetween a desirable and undesirable analytes in a sample. A specificbinding agent typically differentiates between a desirable andundesirable analyte by binding more than about 10 to 100-fold or more toa desirable analyte in preference to other analytes. In certaincircumstances, such preferential binding can be at least 1000- to10,000-fold. For example, the affinity between a capture agent andanalyte when they are specifically bound in a capture agent/analytecomplex is at least about 10⁻⁷ M, at least about 10⁻⁸ M, at least about10⁻⁹ M, at least about 10⁻¹⁰ M, or about at least 10⁻¹¹ e.g., at least10⁻⁷ M, at least 10⁻⁸ M, at least 10⁻⁹ M, at least 10⁻¹⁰ M, or at least10⁻¹¹.

As used herein, the term “capture agent/analyte complex” is a complexthat results from the specific binding of a capture agent with ananalyte.

As used herein, “binding partners” refers to pairs of molecules that canbe found in a capture agent/analyte complex, i.e., exhibit specificbinding with each other.

As used herein, the term “assessing” refers to any form of measurement,and includes determining if an element is present or not. The terms“determining”, “measuring”, “evaluating”, “assessing”, and “assaying”are used interchangeably and include both quantitative and qualitativedeterminations.

As used herein, a “detectable label” refers to an atom (e.g.,radionuclide), molecule (e.g., fluorescein), or complex, that isdetected or can be used to detect (e.g., due to a physical or chemicalproperty) the presence of an analyte, or to enable binding of anothermolecule to which it is covalently bound or otherwise associated. Theterm also refers to covalently bound or otherwise associated molecules(e.g., a biomolecule such as an enzyme) that act on a substrate toproduce a detectable atom, molecule or complex. Detectable labelsinclude any composition detectable by spectroscopic, photochemical,biochemical, immunochemical, electrical, optical or chemical means. Forexample, labels include biotin for staining with labeled streptavidinconjugate, magnetic beads (e.g., Dynabeads™), fluorescent dyes (e.g.,fluorescein, Texas red, rhodamine, green fluorescent protein, enhancedgreen fluorescent protein, and the like), radiolabels (e.g., ³H, ¹²⁵I,³⁵S, ¹⁴C, or ³²P), enzymes (e.g., hydrolases, particularly phosphatasessuch as alkaline phosphatase, esterases and glycosidases, oroxidoreductases, particularly peroxidases such as horse radishperoxidase, and others commonly used in ELISAs), substrates, cofactors,inhibitors, chemiluminescent groups, chromogenic agents, andcolorimetric labels such as colloidal metallic particles (such as gold),colloidal non-metallic particles (such as selenium or tellurium), orcolored glass or plastic (e.g., polystyrene, polypropylene, latex, etc.)beads. Non-limiting examples of patents disclosing such labels includeU.S. Pat. Nos. 3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437;4,275,149; and 4,366,241, each of which is incorporated by reference inits entirety. Means of detecting such labels are known to those of skillin the art.

As used herein, the terms “sandwich”, “sandwich ELISA”, “sandwichdiagnostic”, and “capture ELISA” refer to the concept of detecting ananalyte with two or more different test agents. For example, a captureagent can be directly or indirectly attached to a substrate, and a testsample can be passed over the surface of the substrate allowing thecapture agent to specifically bind its cognate analyte. A labeledreagent, e.g., a labeled antibody or alternative detection reagent(which can bind the analyte), can then be used to determine whether acapture agent specifically bound the analyte.

As used herein, the term “substrate” means any support capable ofbinding or being associated with a capture agent. Well-known substratesinclude glass, polystyrene, polypropylene, polyethylene, dextran, nylon,amylases, natural and modified celluloses, polyacrylamides, agarose, andmagnetite. The nature of the substrate can be either soluble to someextent or insoluble. The structure of the substrate is not limiting, andthe substrate can have any structural configuration so long as thecapture agent is capable of binding to an analyte. Thus, the structuralconfiguration can be spherical, as in a bead, or cylindrical, as in theinside surface of a test tube, or the external surface of a rod.Alternatively, the surface can be flat, such as a sheet, culture dish,test strip, etc. Those skilled in the art will know many other suitablesubstrates for binding or being associated with a capture agent, or canascertain the same by routine experimentation.

As used herein, the term “subject” refers to any animal, e.g., a mammal,human or non-human. Exemplary subjects include, but are not limited to,humans, non-human primates, mice, rats, guinea pigs, cattle, sheep,goats, pigs, dogs, cats, birds, deer, elk, rabbit, reindeer, deer, andhorses.

As used herein, the term “measuring the level of” refers to detectionboth quantitatively and qualitatively.

The following figures are presented for the purpose of illustrationonly, and are not intended to be limiting.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic of the lateral flow apparatus and the results of atest corresponding to Example 1 following the loading of variouscomponents.

FIG. 2 is a schematic of the lateral flow apparatus and the results of atest corresponding to Example 2 following the loading of variouscomponents.

FIG. 3 is a schematic of the lateral flow apparatus and the results of atest corresponding to Example 3 following the loading of variouscomponents.

FIG. 4 is a schematic of the lateral flow apparatus and the results of atest corresponding to Example 4 following the loading of variouscomponents.

Other features and advantages of the invention will be apparent from thefollowing detailed description, and from the claims.

DETAILED DESCRIPTION

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention, suitable methods andmaterials are described below. All publications, patent applications,patents, and other references mentioned herein, including GenBankdatabase sequences, are incorporated by reference in their entirety. Incase of conflict, the present specification, including definitions, willcontrol. In addition, the materials, methods, and examples areillustrative only and not intended to be limiting.

Patients receiving drug therapies that are immuno-modulatory arevulnerable to certain diseases such as progressive multifocalleukoencephalopathy (PML). For example, PML has been reported inpatients treated with natalizumab. It has been found that one method oftreating such subjects is to reduce the amount (e.g., plasmaconcentration) of the immuno-modulatory therapeutic agent (e.g.,natalizumab) in the patient. Because of the rapid progression of thedisease and the wide geographic distribution of patients, as well asother issues, there is a need for a rapid, reliable test that can beused to determine the level of the drug in the patient. It is anadvantage for the test to be formatted so it can be administered andaccurately interpreted within a short period of time. This is aparticular issue with an immuno-modulatory therapy, which may bedifficult to distinguish from a patient's own antibodies. Accordingly,this problem has been solved by using a rapid test design that canaccurately and easily be used by physicians to determine whether thelevel of an immuno-modulatory therapy has been sufficiently modulated toincrease the likelihood of mitigating the progression of an active caseof PML. By varying the dosage of the immuno-modulatory therapy, theprogression of a patient's disease state can be alleviated. Becausevariance of the dosage of the immuno-modulatory therapy is important fortreatment, assays described herein can also be used to rapidly monitordrug level in a patient, for example, in multiple sclerosis patientsincluding pediatric patients. In another embodiment, assays providedherein can detect anti-integrin antibodies in a subject who was treatedwith a natalizumab and has been diagnosed with or is suspected of havinga JC virus infection. The JC virus is a type of human polyomavirus whichcan cause PML and other diseases in patients with immunodeficiency or inpatients treated with drug intended to induce a state ofimmnosuppression (e.g. organ transplant patients).

In addition, assays are provided herein to detect anti-drug antibodies.The presence of such antibodies in a subject may impact drug exposureand/or result in infusion related adverse events, for example, as hasbeen reported upon treatment with a humanized anti-VLA-4 antibody.Therefore, assays for the detection of anti-drug antibodies (e.g., thedrug being the anti-VLA-4 antibody; the anti-drug antibodies beinganti-anti-VLA-4 antibodies) can be useful for treatment using the drugof interest (e.g., an anti-VLA-4 drug such as a natalizumab).

In general, the apparatus is a solid substrate associated with a captureagent that can be used to specifically detect the immuno-modulatorytherapeutic agent (e.g., natalizumab) or antibodies against animmuno-modulatory agent (e.g., anti-drug antibody such as antibodiesagainst natalizumab). In some cases, the detection is calibrated toprovide a method of determining whether the amount of immuno-modulatorytherapeutic agent or anti-drug antibody is above or below a specifiedconcentration or titer. Such information can be correlated with whetheradditional therapy (e.g., plasmapheresis) is needed to further reducethe amount of the immuno-modulatory therapeutic agent in the patient,thereby mitigating the risk associated with PML.

Also provided herein is a kit suitable for testing for the presence ofan immuno-modulatory therapeutic agent such as natalizumab, orantibodies against such an immuno-modulatory therapeutic reagent. A kitwill generally include at least one reagent or apparatus as describedherein for detecting the immuno-modulatory therapeutic agent orantibodies against an immuno-modulatory agent. The kit can also includeinstructions for use of the reagents and/or apparatus. Such instructionscan also include use of the kit for determining an amount of an analyteand interpretation of assay results. For example, the instructions canprovide guidance as to whether detection of a specified level of anatalizumab is sufficient to treat a subject having or suspected ofhaving PML.

Apparatus for Assaying Analytes

Various analytical devices are known to those of skill in the art, andsuch analytical devices can be adapted to utilize one or more captureagents, e.g., an antibody described herein, to assay one or moreanalytes, e.g., an anti-integrin antibody such as an anti-VLA-4 (e.g., anatalizumab), e.g., an anti-drug antibody (e.g., anti-natalizumab)described herein. Nonlimiting examples include dipstick or tester,lateral flow, dual flow and flow-through devices, particularly thosethat are immunoassays. Non-limiting examples of lateral flow devicesinclude those described in U.S. Pat. Nos. 4,818,677, 4,943,522,5,096,837 (RE 35,306), 5,096,837, 5,118,428, 5,118,630, 5,221,616,5,223,220, 5,225,328, 5,415,994, 5,434,057, 5,521,102, 5,536,646,5,541,069, 5,686,315, 5,763,262, 5,766,961, 5,770,460, 5,773,234,5,786,220, 5,804,452, 5,814,455, 5,939,331, 6,306,642, each of which isincorporated by reference in its entirety. Other non-limiting examplesof lateral flow devices include U.S. Pat. Nos. 4,703,017, 6,187,598,6,352,862, 6,485,982, 6,534,320 and 6,767,714, each of which isincorporated by reference in its entirety. Non-limiting examples ofdipstick devices include those described in U.S. Pat. Nos. 4,235,601,5,559,041, 5,712,172 and 6,790,611, each of which is incorporated byreference in its entirety.

An apparatus for assaying the level of an analyte can include asubstrate, e.g., a substrate with which a capture agent is associated.Non-limiting examples of substrates include glass, polystyrene,polypropylene, polyethylene, dextran, nylon, amylases, natural andmodified celluloses, polyacrylamides, agarose, and magnetite. Thesubstrate can have any structural configuration so long as the captureagent is capable of binding to an analyte.

In some instances, the substrate is housed in a support, e.g., a supportthat is spherical, as in a bead, or cylindrical, as in the insidesurface of a test tube, or the external surface of a rod. Alternatively,the substrate or support can be flat, such as a sheet, culture dish,test strip, and the like.

In one example, the apparatus is a lateral flow apparatus that includesa sample receiving zone, a label zone, a test zone and a control zone.In some instances, a test region comprises the test and control zones.In some instances, the test region, which comprises the test and controlzones, is observable.

In certain examples, the sample receiving zone accepts a fluid samplethat may contain one or more analytes of interest. In another example,the sample receiving zone is dipped into a fluid sample. A label zone islocated downstream of the sample receiving zone, and contains one ormore mobile labeled reagents that recognize, or are capable of binding,one or more analytes of interest. Further, a test region is disposeddownstream from the label zone, and contains test and control zones. Thetest zone(s) generally contain a capture agent associated with thesubstrate at the test zone. In certain instances, the capture agent isimmobilized on the substrate at the test zone. Generally, theimmobilized capture agent specifically binds to the analyte of interest.

As the fluid sample flows along the substrate, the analyte of interestwill first bind with a mobilizable labeled reagent in the label zone,and then become restrained in the test zone. In some examples, the testregion is comprised of a material that is opaque in a dry state andtransparent in a moist state. Thus, when a control zone comprising amark on the device is utilized, this mark is positioned about the testregion such that it becomes visible within the test region when the testregion is in a moist state.

In another instance, the fluid sample flows along a flow path runningfrom the sample receiving zone (upstream), through the label zone, andthen to the test and control zones (together comprised in a test region)(downstream). Optionally, the fluid sample may thereafter continue tothe absorbent zone.

In another example, the sample receiving zone is composed of anabsorbent application pad. Suitable materials for manufacturingabsorbent application pads include, but are not limited to, hydrophilicpolyethylene materials or pads, acrylic fiber, glass fiber, filter paperor pads, desiccated paper, paper pulp, fabric, and the like. Forexample, the sample receiving zone may be composed of a material such asa nonwoven spunlaced acrylic fiber (e.g., available from DuPontNonwovens) or HDK material (available from HDK Industries, Inc.,Rogersville, Tenn.). In another example, the sample receiving zone isconstructed from any material that is capable of absorbing water.

In other examples, the test device is configured to perform animmunological assay. In some instances, the liquid transport along thesubstrate is based upon capillary action. In another situation, theliquid transport along the substrate is based on non-bibulous lateralflow, wherein all of the dissolved or dispersed components of the liquidsample are carried at substantially equal rates and with relativelyunimpaired flow laterally through the substrate, as opposed topreferential retention of one or more components as would occur, e.g.,in materials that interact, chemically, physically, ionically orotherwise with one or more components (see, for example, U.S. Pat. No.4,943,522, which is incorporated by reference in its entirety).

The labeling zone of immunoassay devices can also include control-typereagents. These labeled control reagents often comprise detectablemoieties that will not become restrained in the test zones and that arecarried through to the test region and control zone(s) by fluid sampleflow through the device. In some instances, these detectable moietiesare coupled to a member of a specific binding pair to form a controlconjugate that can then be restrained in a separate control zone of thetest region by a corresponding member of the specific binding pair toverify that the flow of liquid is as expected. The visible moieties usedin the labeled control reagents can be the same or different color, orof the same or different type, as those used in the analyte of interestspecific labeled reagents.

The test region generally includes a control zone that is useful toverify that the sample flow is as expected. Each of the control zonescan comprise a spatially distinct region that can include an immobilizedmember of a specific binding pair that reacts with a labeled controlreagent. In some examples, the procedural control zone contains anauthentic sample of the analyte of interest, or a fragment thereof. Inthis instance, one type of labeled reagent can be utilized, whereinfluid sample transports the labeled reagent to the test and controlzones; and the labeled reagent not bound to an analyte of interest willthen bind to the authentic sample of the analyte of interest positionedin the control zone. In another example, the control line containsantibody that is specific for, or otherwise provides for, theimmobilization of the labeled reagent. In operation, a labeled reagentis restrained in each of the one or more control zones, even when any orall the analytes of interest are absent from the test sample.

Capture Agents

Various capture agents can be used in the methods and apparatusdescribed herein, provided the capture agent specifically binds ananalyte of interest to form a capture agent/analyte complex. In certaininstances, the capture agent is an antigen, e.g., an integrin, and theanalyte of interest is an antibody, e.g., an anti-integrin antibody oran alpha 4 integrin binding antibody (e.g. natalizumab) or an anti-drugantibody, e.g., anti-anti-VLA4 integrin chain antibody.

In some situations, the capture agent is a polypeptide, e.g., anantibody, that binds to an analyte of interest. Any antibody thatspecifically binds to an analyte can be utilized as a capture agent. Forexample, the antibody can be a polyclonal antibody; a monoclonalantibody or antigen binding fragment thereof; a modified antibody suchas a chimeric antibody, reshaped antibody, humanized antibody, orfragment thereof (e.g., Fab′, Fab, F(ab′)₂); or a biosynthetic antibody,e.g., a single chain antibody, single domain antibody (DAB), Fv, singlechain Fv (scFv), or the like.

Methods of making and using polyclonal and monoclonal antibodies aredescribed, e.g., in Harlow et al., Using Antibodies: A LaboratoryManual: Portable Protocol I. Cold Spring Harbor Laboratory (Dec. 1,1998), which is incorporated by reference in its entirety. Methods formaking modified antibodies and antibody fragments (e.g., chimericantibodies, reshaped antibodies, humanized antibodies, or fragmentsthereof, e.g., Fab′, Fab, F(ab′)₂ fragments); or biosynthetic antibodies(e.g., single chain antibodies, single domain antibodies (DABs), Fv,single chain Fv (scFv), and the like), are known in the art and can befound, e.g., in Zola, Monoclonal Antibodies: Preparation and Use ofMonoclonal Antibodies and Engineered Antibody Derivatives, SpringerVerlag (Dec. 15, 2000; 1st edition), which is incorporated by referencein its entirety.

In particular instances, the capture agent is an antibody that canspecifically bind an anti-integrin antibody (e.g., an anti anti-VLA-4antibody such as an anti-natalizumab), such as the rat 12C2 antibody,murine 12C4 antibody, or a suitable rabbit antibody. In anotherembodiment, the capture agent is an anti-VLA-4 antibody, such asnatalizumab, that can specifically bind to an anti-(anti-VLA-4 antibody)antibody.

Analytes

The methods and apparatus described herein can be used to assay thelevel of an analyte of interest, e.g., in a biological sample from asubject. Exemplary analytes include, without limitation, toxins, organiccompounds, polypeptides, microorganisms, bacteria, viruses, amino acids,nucleic acids, carbohydrates, hormones, steroids, vitamins, drugs(including those administered for therapeutic purposes as well as thoseadministered for illicit purposes), pollutants, pesticides, andmetabolites of or antibodies to any of the above substances. Anyantigenic substance, hapten, antibody, macromolecule, and combinationsthereof, can also be an analyte assayed using a method or an apparatusdescribed herein. In particular instances, the analyte is an antibody,such as an anti-integrin antibody.

Integrins are known in the art (see, e.g., Takeda et al., Genome Biol.8:215 (2007)), which is incorporated by reference in its entirety, andthe level of any anti-integrin antibody can be assayed using a method oran apparatus described herein. An antibody can be, e.g., a polyclonalantibody; a monoclonal antibody or antigen binding fragment thereof; amodified antibody such as a chimeric antibody, reshaped antibody,humanized antibody, or fragment thereof (e.g., Fab′, Fab, F(ab′)₂); or abiosynthetic antibody, e.g., a single chain antibody, single domainantibody (DAB), Fv, single chain Fv (scFv), or the like. The antibodycan be naturally produced by the subject or one that is administered tothe subject.

In some instances, the analyte is an anti-VLA-4 antibody, e.g., ahumanized anti-VLA-4 antibody, for example a natalizumab. TYSABRI® andother biologically active analogues, variants or derivatives ofTYSABRI®, including biosimilars and biologics substantially similarthereto, qualify as a natalizumab. In particular situations, the analyteis an anti-VLA-4 antibody previously administered to the subject, e.g.,as a therapeutic antibody. The analyte can be, for example, anatalizumab. In another situation, the analyte is an anti-(anti-VLA-4antibody) antibody. The analyte can be, for example, anti-natalizumabantibody.

Biological Samples

Biological samples to be assayed for the presence of analyte using themethods and apparatus described herein can be obtained from any subject,e.g., a human or a non-human subject. In some embodiments, thebiological sample is obtained from a living human subject.

In some instances, the subject from whom the sample is obtained isapparently healthy, where the assay is performed as a part of routinescreening. In other embodiments, the subject has been provisionallydiagnosed as having a disease or disorder and has undergone treatment,e.g., with an anti-integrin antibody, e.g., an anti-VLA-4 antibody. Thebiological sample can be from a subject who has been treated to decreasethe amount of analyte. In some cases, a sample from a subject is testedusing a method described herein before the subject undergoes atreatment, e.g., to modulate the amount of analyte in the subject or toestablish the baseline level of analyte in the subject or to establishthe suitability of the treatment, and after the subject has beentreated, e.g., to monitor and/or to modulate the amount of analyte inthe subject.

The biological sample can be derived from any tissue, organ or group ofcells of the subject. In some circumstances, the biological sample is acervical scrape, biopsy, or lavage obtained from a subject. In otherinstances, the sample is a blood, plasma, serum, or urine sample.

In some situations, the biological sample can be processed, e.g., toremove certain components that may interfere with a method describedherein, using methods that are known in the art. For example, thebiological sample can be processed to be enriched for proteins, e.g., bysalt precipitation, and the like.

In certain methods described herein, the level of an analyte in a samplecan be quantified and/or compared to controls. Suitable control samplescan be, e.g., from individuals who have not received a treatment, e.g.,an anti-integrin antibody treatment, e.g., an anti-VLA-4 antibodytreatment. Control samples can be from individuals genetically relatedto the subject being assayed, but can also be from genetically unrelatedindividuals. In some cases, the control is an established reference. Inother instances, the level of an analyte, e.g., an anti-integrinantibody, is quantified using known methods. In yet other situations,the presence or absence of an analyte, e.g., an anti-integrin antibody,within a sample is determined by visual inspection of the assay.

In certain embodiments, a sample is contacted to a solid support-boundcapture agent under conditions suitable for binding of the capture agentto analytes in the sample, and, after separation of unbound sampleanalytes from the bound analytes, the bound analytes are detected asdescribed herein.

Methods of Assaying

In some situations, an analyte, e.g., an anti-integrin antibody, e.g.,an anti-VLA-4 antibody, or e.g., an anti-(anti-VLA-4 antibody) antibody,can be assayed using a method selected from a variety of immunoassaymethods, both qualitatively and quantitatively. Immunological andimmunoassay procedures are known and described in, e.g., Stites et al.(eds.) Basic and Clinical Immunology (4th ed.) Lange MedicalPublications, Los Altos, Calif., which is incorporated by reference inits entirety. Immunoassays can be performed in any of severalconfigurations known in the art and described in, e.g., Maggio EnzymeImmunoassay, CRC Press, Boca Raton, Fla. (1980); Tijssen, “Practice andTheory of Enzyme Immunoassays,” Laboratory Techniques in Biochemistryand Molecular Biology, Burdon and van Knippenberg Eds., Elsevier (1985),pp 9-20; and Harlow and Lane, Antibodies, A Laboratory Manual, ColdSpring Harbor Publications, NY (1988), each of which is incorporated byreference in its entirety.

Immunological Binding Assays

In some instances, an analyte, e.g., an anti-integrin antibody, or e.g.,an anti-drug antibody, is detected and/or quantified using any of anumber of known immunological binding assays (see, e.g., U.S. Pat. Nos.4,366,241; 4,376,110; 4,517,288; 4,837,168; and Asai, Methods in CellBiology Volume 37: Antibodies in Cell Biology, Academic Press, Inc. NY(1993), each of which is incorporated by reference in its entirety).

Immunoassays can also utilize a labeled reagent to specifically bind toand label the capture agent/analyte complex. The labeled reagent mayitself be one of the moieties comprising the capture agent/analytecomplex. Alternatively, the labeled reagent may be a third moiety, suchas an antibody, that specifically binds to the analyte or the captureagent/analyte complex.

In some situations, the analyte is an antibody, e.g., an anti-integrinantibody, or e.g., an anti-drug antibody, and the labeled reagent is asecond antibody bearing a label. Alternatively, the second antibody maylack a label, but it may, in turn, be bound by a labeled reagent thatincludes a third antibody specific to antibodies of the species fromwhich the second antibody is derived. The second antibody can bemodified with a detectable moiety, such as biotin, to which a labeledreagent can specifically bind, such as enzyme-labeled streptavidin.

Other proteins that can specifically bind immunoglobulin constantregions, such as protein A or protein G, can also be used as the labeledreagent. These proteins are normal constituents of the cell walls ofstreptococcal bacteria. They exhibit a strong non-immunogenic reactivitywith immunoglobulin constant regions from a variety of species (see,e.g., Kronval et al., J. Immunol. 111:1401-1406 (1973); and Akerstrom etal., J. Immunol. 135:2589-2542 (1985), which is incorporated byreference in its entirety).

Throughout the methods and assays described herein, incubation and/orwashing steps may be required after one or more combination of reagents.Incubations carried out during the assay can vary from about 5 secondsto several hours, in general, from about 5 minutes to about 24 hours.The incubation time will depend upon the assay format, analyte, volumeof solution, concentrations, and the like. In general, an assay iscarried out at ambient temperature, although an assay can be conductedover a range of temperatures, such as about 4° C. to about 40° C., e.g.,4° C.-10° C., or 4° C.-25° C.

1. Non-Competitive Assay Formats

Immunoassays for detecting an analyte of interest, e.g., ananti-integrin antibody, or e.g., an anti-drug antibody, from samples canbe competitive or noncompetitive. Noncompetitive immunoassays are assaysin which the amount of analyte in a capture agent/analyte complex isdirectly measured. In a “sandwich” assay, for example, the capture agentcan be bound directly to a solid substrate where it is immobilized.These immobilized capture agents can then specifically bind an analytepresent in a test sample. The analyte, e.g., anti-integrin antibody, ore.g., an anti-drug antibody, thus immobilized is then bound by a labeledreagent, such as a second antibody bearing a label. Alternatively, thesecond antibody may lack a label, but it may, in turn, be bound by alabeled reagent that is a third antibody specific to antibodies of thespecies from which the second antibody is derived. The second antibodycan be modified with a detectable moiety, such as biotin, to which athird labeled reagent can specifically bind, such as enzyme-labeledstreptavidin.

2. Competitive Assay Formats

In competitive assays, the amount of analyte (such as an anti-integrinantibody or an anti-drug antibody) present in a sample is measuredindirectly by measuring the amount of an added (exogenous) analytedisplaced (or competed away) from a capture agent (e.g., an antibodyspecific for the analyte) by the analyte present in the sample. In onecompetitive assay, a known amount of an analyte, e.g., anti-integrinantibody, or e.g., an anti-drug antibody, is added to the sample and thesample is then contacted with a capture agent, e.g., an antibody thatspecifically binds to an anti-integrin antibody: The amount of analytebound to the capture agent, e.g., antibody, is inversely proportional tothe concentration of analyte present in the sample. In a particularexample, the capture agent, e.g., an antibody that binds to ananti-integrin antibody, is immobilized on a solid substrate. Forexample, the amount of the analyte, e.g., anti-integrin antibody, ore.g., an anti-drug antibody, bound to the capture agent, e.g., antibodythat binds an anti-integrin antibody, or e.g., drug that anti-drugantibody binds to, may be determined either by measuring the amount ofanalyte present in a capture agent/analyte complex or, alternatively, bymeasuring the amount of remaining uncomplexed analyte. The amount ofanalyte may be detected by providing a labeled reagent.

3. Other Assay Formats

In other instances, when the analyte is a polypeptide, western blot(immunoblot) analysis can be used to detect and quantify the presence ofthe polypeptide in a sample. The technique generally comprisesseparating sample proteins by gel electrophoresis on the basis ofmolecular weight, transferring the separated proteins to a suitablesolid support (such as, e.g., a nitrocellulose filter, a nylon filter,or a derivatized nylon filter), and incubating the sample with theantibodies that specifically bind the polypeptide of interest. Forexample, the antibodies specifically bind to a polypeptide of intereston the solid support. These antibodies may be directly labeled oralternatively may be subsequently detected using labeled antibodies(e.g., labeled sheep anti-mouse antibodies) that specifically bind tothe antibodies against the polypeptide of interest.

Other assay formats include liposome immunoassays (LIA), which useliposomes designed to bind specific molecules (e.g., antibodies) andrelease encapsulated reagents or markers. The released chemicals arethen detected according to standard techniques (see, e.g., Monroe etal., Amer. Clin. Prod. Rev. 5:34-41(1986), which is incorporated byreference in its entirety).

4. Labeled Reagents

The detectable label used in a method or apparatus described herein isnot limiting as long as it does not significantly interfere with thespecific binding of the labeled reagent used in the method or apparatus.The detectable label can be any material having a detectable physical orchemical property. Such detectable labels have been well developed inthe field of immunoassays and, in general, most labels useful in suchmethods can be used in the methods and apparatus described herein. Alabel can be, e.g., any composition detectable by spectroscopic,photochemical, biochemical, immunochemical, electrical, optical orchemical means. Useful labels in the methods and apparatus describedherein include, without limitation, magnetic beads (e.g., Dynabeads™),fluorescent dyes (e.g., fluorescein isothiocyanate, Texas red,rhodamine, and the like), radiolabels (e.g., ³H, ¹²⁵I, ³⁵S, ¹⁴C, or³²P), enzymes (e.g., horseradish peroxidase, alkaline phosphatase andothers that can be used in an ELISA), and colorimetric or particulatelabels such as colloidal gold or colored glass or plastic (e.g.,polystyrene, polypropylene, latex, etc.) beads.

The label can be coupled directly or indirectly to the desired componentof the assay according to methods known in the art. As described herein,a wide variety of labels can be used, with the choice of label dependingon the sensitivity required, the ease of conjugation, stabilityrequirements, available instrumentation, and disposal provisions.

Non-radioactive labels can be attached by indirect means. Molecules canalso be conjugated directly to signal generating compounds, e.g., byconjugation with an enzyme or fluorescent compound. A variety of enzymesand fluorescent compounds can be used with the methods and apparatusdescribed herein and are well-known to those of skill in the art (see,e.g., U.S. Pat. No. 4,391,904, which is incorporated by reference in itsentirety).

Means of detecting labels are known to those of skill in the art. Thus,for example, where the label is a radioactive label, means for detectioninclude a scintillation counter or photographic film as inautoradiography. Where the label is a fluorescent label, it can bedetected by exciting the fluorochrome with the appropriate wavelength oflight and detecting the resulting fluorescence. The fluorescence can bedetected visually, by means of photographic film, by the use ofelectronic detectors such as charge-coupled devices (CCDs) orphotomultipliers and the like. Similarly, enzymatic labels can bedetected by providing the appropriate substrates for the enzyme anddetecting the resulting reaction product. Finally simple colorimetriclabels can be detected directly by observing the color associated withthe label. Thus, in various dipstick assays, conjugated gold oftenappears pink, while various conjugated beads appear the color of thebead.

Some assay formats do not require the use of labeled reagents. Forinstance, agglutination assays can be used to detect the presence oftarget antibodies. In this case, antigen-coated particles areagglutinated by samples comprising target antibodies. In this format,none of the assay components need to be labeled, and the presence of thetarget antibody is detected by simple visual inspection.

After detection of a detectable signal, the signal can be compared to areference and/or correlated with a predetermined level corresponding totreatment recommendations. For example, in an assay to detect the levelof an anti-VLA-4 antibody in a subject being treated with plasmapheresisto reduce the amount of the antibody, a level of signal below apredetermined level may be correlated with a recommendation that furtherplasmapheresis is not required. A signal correlated with a higher levelmay indicate that additional plasmapheresis is advisable.

Detection of signal in an assay described herein can be visual, but mayalso be performed using a reader to detect a signal. Such readersinclude, for example, automated plate readers, EIA readers, and thelike. Readers can be used for semi-quantitative or quantitativedetermination of the concentration for tested analytes.Semi-quantitative or quantitative determination of concentration can bedepicted using a colorimetric system.

5. Secondary Agents

The secondary agents used in a method or apparatus described herein canbe used to detect binding of the capture agent to the antibody, Thesecondary agent can be an antibody that has a detectable label attached,either covalently or non-covalently. The detectable label attached tothe secondary agent can be a detectable label as previously described.The secondary agent can be a non-specific antibody, or antibodies thatrecognize the analyte or fragments thereof or polypeptides thatrecognize the analyte. The secondary agent can include anti-IgG4antibody, for example, anti-human IgG4 or anti-human IgG4 that reactswith the Fc portion of the heavy chain of human IgG4.

6. Time

Binding of the capture agent to the antibody can be detectable withinabout 30 seconds, 1 minute, 5 minutes, 10 minutes, 20 minutes, 30minutes, 40 minutes, 50 minutes, 1 hour, 2 hours, 5 hours, or 10 hoursafter the binding.

7. Buffer

Any suitable buffer for immunological assays can be used as a chasebuffer as determined by a person of skill in the art. The chase buffercan include a surfactant. Any suitable surfactant can be used. Asurfactant can be a polysorbate surfactant such as TWEEN 20 ®, TWEEN 40®, TWEEN 60 ®, TWEEN 80 ®, SPAN 20 ®, SPAN 40 ®, SPAN 60 ®, SPAN 65 ®,and SPAN 80 ®. The buffer can also include a buffered saline, such asTris buffered saline (TBS) or phosphate buffered saline (PBS). Thebuffer can also include a blocking agent, for example a protein such asbovine serum albumin (BSA), milk or gelatin.

Treatments

The assays described herein can be performed before and/or afteradministering a treatment to a subject. In some instances, the treatmentis a treatment to remove an analyte or component from the subject, suchas from the blood of a subject. In particular situations, the treatmentis blood apheresis, such as plasma exchange, to remove one or moreantibodies from the subject.

Blood apheresis is a common medical use of continuous fluid separation.Apheresis has many clinical uses, including multiple therapies thatinvolve removing blood from a subject's body, separating the blood intocomponents, altering one or more of the components, and putting somemixture or selection from the removed and/or altered fluid back into thesubject's body. Some exemplary therapeutic apheresis procedures includetherapeutic plasma exchange (TPE or PLEX) (a procedure by whichcell-free plasma is removed and replaced with colloid/saline solution);cytoreduction (a process by which platelets and white blood cells areremoved); photopheresis (a procedure by which mononuclear cellscollected by therapeutic apheresis are exposed to ultraviolet-A lightand psoralen, and reinfused into the subject); and selective adsorption(a process by which plasma is adsorbed on a column and returned to thesubject).

In one exemplary apheresis procedure, blood is withdrawn from a subjectthrough a needle inserted into the vein of the subject. The needle isattached to one end of a plastic tube that provides a flow path for theblood. The other end of the tube leads to a separator, such as acentrifuge, for separating the blood into its components. Flow-throughcentrifuges that allow for the continuous inflow and outflow ofmaterials to and from the centrifuge are well known in the art (see, forexample, U.S. Pat. No. 4,425,112, which is incorporated by reference inits entirety). The blood that is separated into plasma and cells can bereturned to the subject through the other arm. A plasma substitute or analtered plasma can be recombined with the blood elements to be returnedto the subject. Since the rate of blood being withdrawn from the subjectand the blood returning to the subject can be at the same rate, only asmall amount of the subject's blood can be outside of the body at anytime.

Another apheresis procedure utilizes an automated system that usesdisposable, pre-sterilized fluid circuits through which the blood flows.The fluid circuits are mounted on reusable machines that may have pumps,valves, sensors, and the like. These automated systems further includean internal computer and associated software programs that control manyof the processing functions. One exemplary automated system is describedin U.S. Pat. No. 6,706,008, which is incorporated by reference in itsentirety. Other methods and apparatus for apheresis are described in,e.g., U.S. Pat. Nos. 7,267,771; 6,849,183; 5,200,090; and 4,954,128,each of which is incorporated by reference in its entirety.

In some situations, the level of an anti-integrin antibody or ananti-drug antibody described herein is assayed before administering atreatment to a subject. In other situations, the level of ananti-integrin antibody or an anti-drug antibody is assayed afteradministering a treatment to a subject. For example, the level of ananti-integrin antibody or anti-drug antibody is assayed one day, twodays, three days, four days, five days, six days, one week, two weeks,three weeks, or four weeks after the administration of the treatment.Such assays can be administered as part of a treatment regime, forexample to periodically monitor drug levels or levels of anti-drugantibodies. In certain instances, the treatment is administered and thelevel of an anti-integrin antibody or anti-drug antibody assayed inrepeated cycles of treatment and assay, for example, until the level ofthe anti-integrin antibody in the sample reaches a predetermined level.For example, the predetermined level can be about 1 μg/ml, about 2μg/ml, about 3 μg/ml, about 4 μg/ml, about 5 μg/ml, about 6 μg/ml, about7 μg/ml, about 8 μg/ml, about 9 μg/ml, about 10 μg/ml, about 15 μg/ml,or about 20 μg/ml. In some cases, the predetermined level is 1 μg/ml, 2μg/ml, 3 μg/ml, 4 μg/ml, 5 μg/ml, 6 μg/ml, 7 μg/ml, 8 μg/ml, 9 μg/ml, 10μg/ml, 15 μg/ml, or 20 μg/ml. The treatments can be administered, e.g.,once a day, or once every two days, three days, four days, five days,six days, one week, two weeks, three weeks, four weeks, every threemonths, every four months, every six months, or annually.

The invention is further illustrated by the following examples. Theexamples are provided for illustrative purposes only. They are not to beconstrued as limiting the scope or content of the invention in any way.

EXAMPLES Example 1 Development of a Test to Assay Anti-VLA-4 Antibody inBuffer Using Rat 12C2 mAb or Mouse 12C4 mAb and Anti-Human IgG4-GoldConjugates

A lateral flow immunochromatographic test system was used to develop ananti-VLA-4 specific test. Referring to FIG. 1, the lateral flow testsystem was composed of a two part plastic cassette (top 100 and bottom101). The top part contained two openings—one opening 102 for theapplication of samples 110 and chase buffer 118 and a window 103 forvisual detection of the results. The lower part accommodated anitrocellulose test strip 104 on which the rat 12C2 mAb or mouse 12C4mAb 105 was immobilized at a “test site” (“T”) 106 and a universal goldcapture reagent 107 at a “control site” (“C”) 108.

Upstream from the test and control sites, there was a “gold pad” 109 offibrous material where a mouse anti-human IgG4 was conjugated to goldparticles, the complex being 112. Further upstream from the goldconjugate pad was another fibrous pad 111 that received the samplehumanized anti-VLA-4 antibody in buffer (110) and chase buffer 118 viaopening 102. The sample pad 111, gold pad 109, and nitrocellulosemembrane 104 were in sequential order so that the sample migrated bycapillary action from the sample pad 111 to the gold pad 109 to form a“first complex” of sample 110 and mouse anti-human IgG4/gold conjugate112. The complex then traveled through the nitrocellulose 104 where itencountered the mouse 12C4 mAb or rat 12C2 mAb 105 at the test site 106.The sample 110 reacted with the mouse 12C4 mAb or rat 12C2 mAb 105,forming a complex of 112 and 110 that was arrested at the test site 106and was visualized as a red line 114. In another embodiment, the sample110 can be added downstream of the gold conjugate.

Excess gold labeled free antibody (mouse anti-human IgG4) traveledfurther to the control site where a universal gold capture reagent 107captured the gold-labeled free antibody (mouse anti-human IgG4), givingrise to an additional visible red line 115 at control site 108. Anyexcess material, gold, and chase buffer was absorbed at the distal endof the nitrocellulose strip by an absorbent filter pad 116.

The specificity of the test was due to the fact that the complex at thetest site only formed when the sample molecule contains a human orhumanized IgG4 moiety to capture the mouse anti-human IgG4 goldconjugate and form the first complex and the anti-VLA-4 moiety to reactwith the anti-(anti-VLA-4 antibody) antibody at the test site. In asimplistic representation, the anti-VLA-4 molecule is sandwiched betweenthe gold labeled mouse anti-human IgG4 and the membrane immobilizedanti-(anti-VLA-4 antibody) antibody.

Materials

Mouse anti-(anti-VLA-4 antibody) mAb 12C4 (Elan), 2.1 mg/mL

Rat anti-(anti-VLA-4 antibody) mAb 12C2 (Biogen Idec), 1.76 mg/mL

Mouse anti-human IgG4 mAb (Southern Biotech), 0.5 mg/mL

Humanized anti-VLA-4 Ab (Biogen Idec), 20 mg/mL

Anti-VLA-1 Ab (Biogen Idec),

Human IgG4 (CalBiochem), 1 mg/mL

Humanized anti-VLA-4 Ab formulation buffer (Biogen Idec)

Universal Gold Capture Reagent

Chase buffer (PBS with 1% w/v BSA and 1% w/v Tween 20)

Gold conjugate (40 nm)

Test Development

The approach for developing the anti-VLA-4 antibody test was to targetthe antigen binding site with a capture antibody specific for theanti-VLA-4 antibody (mAb 12C4 or 12C2) immobilized on the membrane, anda gold labeled detector antibody (anti-human IgG4) directed against theconstant region of the anti-VLA-4 antibody, which comprises a human IgG4molecule.

If the monoclonal antibody 12C4 or 12C2 is highly specific, no othermolecule is captured on the membrane and thus the test is negative.Anti-VLA-1 is a humanized monoclonal antibody to VLA-1. The constantregion of the antibody is human IgG1, while the variable antigen bindingregion is of mouse origin. Because VLA-1 and VLA-4 are closely related,anti-VLA-1 can be used as an additional control of the test system. Suchan inclusion can be used to confirm the specificity of the test system.

Based on these assumptions, a lateral flow immunochromatographic systemwas developed with the mAb 12C4 or 12C2 directed against the humanizedanti-VLA-4 antibody. To determine the appropriate concentration of thecapture antibody, 12C4 or 12C2 was utilized, which was titrated between0.25 and 1.0 mg/mL on separate test strips and tested against thehumanized anti-VLA-4 antibody at 80 μg/mL. The 12C4 or 12C2 antibody wasthen immobilized at the test site of the membrane at 1.0 mg/mL. A mousemonoclonal antibody anti-human IgG4 (gamma chain specific) at 30 μg/mLwas conjugated to gold particles. The mouse anti-human IgG4 immunogoldconjugate was then lyophilized in the conjugate pad and utilized as adetector molecule. At the control site of the membrane, universal goldreagent at 3.0 mg/mL was utilized.

Results

During the titration of the monoclonal antibody 12C4 or 12C2, a positivereaction was indicated by the presence of a band on the top portion ofeach strip. The 1.0 mg/mL antibody concentration was selected, as itresulted in an intense signal with minimum background, and wasimmobilized at the test site of the membrane.

The humanized anti-VLA-4 antibody, humanized anti-VLA-1, IgG1 (myeloma),IgG4 (myeloma), anti-IgG1 and the formulation buffer are tested on aflow system with 12C4 or 12C2 immobilized. The humanized anti-VLA-4antibody was tested at the concentration range of 0.015 and 500 μg/mL.The 12C4 or 12C2 antibody was able to detect the humanized anti-VLA-4antibody to a concentration of 0.08 μg/mL.

The 12C2 antibody was then used to manufacture test cassettes. FIG. 1illustrates the specificity of the test utilizing the test cassettes.All tests showed a single band 115, indicating a valid test, but onlythe cassette for the humanized anti-VLA-4 antibody test showed a secondband 114 indicating a positive test.

Example 2 Development of a Test to Assay Anti-VLA-4 Antibody in SerumUsing Rat 12C2 mAb or Mouse 12C4 mAb and Anti-Human IgG4-Gold Conjugates

To test the viability of the test using a biological fluid, an assay wastested using serum spiked with anti-VLA-4. The two-part plastic cassetteof this lateral flow test system is the same as that for Example 1 witha few alterations. Referring to FIG. 2, the top part 200 contained threeopenings—opening 202 is for the application of samples 210, opening217—for chase buffer 218 and a window 203 for visual detection of theresults. In other embodiments (not shown), the chase buffer can be addeddownstream of the sample or added at the same opening as the one forapplication of samples. The lower part 201 accommodated a nitrocellulosetest strip 204 on which the mouse 12C4 mAb or rat 12C2 mAb 205 wasimmobilized at a “test site” (T) 206 and a goat anti-mouse antibody 207at a “control site” (C) 208. In other embodiments, antibody againstserum-specific analytes (e.g., serum albumin), may be utilized ascontrol antibody.

Upstream from the test and control sites, there was a fibrous pad(“sample pad”) 209 that received the sample 210. Further upstream fromthe test and control sites there was a “gold pad” 211 of fibrousmaterial where a mouse anti-human IgG4 was conjugated to gold particles,the complex being 212. The two-step assay was utilized. In the firststep, serum sample 210 was applied to the sample opening 202 andmigrated toward the capture zone 205 ahead of the gold conjugate to forma complex of a sample 210 (anti-VLA4) with the mouse 12C4 or rat 12C2 ontest line 205. In the second step, the chase buffer 218 was applied tothe chase buffer port 217 rehydrated the monoclonal anti-human IgG4 goldconjugate 212. The conjugate 212 then traveled toward the capture testline 205 to form a complex with anti-VLA-4 bound to capture line 205,and the reaction at site 206 was visualized as a red line 214.

Excess gold-labeled free antibody (mouse anti-human IgG4) traveledfurther to the control site 208 where a goat anti-mouse antibody 207captured the gold-labeled free antibody (mouse anti-human IgG4), givingrise to an additional visible red line 215. Any excess material, gold,and chase buffer was absorbed at the distal end of the nitrocellulosestrip by an absorbent filter pad 216.

The specificity of the test was due to the fact that the complex at thetest site only formed when the sample contains anti-VLA-4 moiety toreact with the anti-(anti-VLA-4 antibody) antibody at the test site, andto be detected by a mouse anti-human IgG4 moiety conjugated to gold. Ina simplistic representation, the anti-VLA-4 molecule is sandwichedbetween the gold labeled mouse anti-human IgG4 and the membraneimmobilized anti-(anti-VLA-4 antibody) antibody. The success of thisformat is reliant upon any endogenous interfering moiety (e.g., IgG4) inthe sample traveling ahead of the anti-IgG4 gold conjugate reducing itsinterference with the detection of the anti-VLA-4 at the test line.

A reader can be used for semi-quantitative determination of theconcentration of the tested analytes. To record the results of thelateral flow system, a reader based on visual positive/negative scoringwas used. The visual threshold on the reader corresponds to valueswithin the 20-40 unit range. If the signal was recorded visuallypositive within this range then it should be considered a very weak pinkline that may not be visible to all operators.

Materials

Mouse anti-(anti-VLA-4 antibody) mAB 12C4 (Elan), 2.1 mg/mL

Mouse anti-human IgG4 mAb (Southern Biotech), 0.5 mg/mL

Humanized anti-VLA-4 Ab (Biogen Idec), 21 mg/mL

Human IgG4 (The Binding Site, San Diego, Calif.), 13.7 mg/mL

Humanized anti-VLA-4 Ab formulation buffer (Biogen Idec)

Goat anti-mouse antibody, 3.0 mg/ml

Chase buffer (PBS with 1% w/v BSA and 1% w/v Tween 20)

Gold conjugate (40 nm)

Test Development

The approach for developing the test was to target the antigen bindingsite with a capture antibody (mAb 12C4 or mAb 12C2) immobilized on themembrane, specific for the anti-VLA-4 antibody, and a gold labeleddetector antibody (mouse anti-human IgG4) directed against the constantregion of the anti-VLA-4 antibody, which comprises a human IgG4molecule.

If a monoclonal antibody used in the assay such as 12C4 or 12C2 ishighly specific, no other analyte is captured on the membrane andtherefore the test is negative.

Based on these assumptions, a lateral flow immunochromatographic systemwas developed with the mAb 12C4 or 12C2 directed against the humanizedanti-VLA-4 antibody. To determine the appropriate concentration of thecapture antibody, 12C4 was utilized, which was titrated on separate teststrips and tested against the humanized anti-VLA-4 antibody at a varietyof concentrations ranging from 100 μg/mL to 0.25 μg/mL. The 12C4antibody was then immobilized at the test site of the membrane at 2.1mg/mL. A mouse monoclonal antibody anti-human IgG4 (gamma chainspecific) was conjugated to 40 nm gold particles. The mouse anti-humanIgG4 immunogold conjugate was then lyophilized in the conjugate pad andutilized as a detector molecule. At the control site of the membrane, agoat anti-mouse antibody at 3.0 mg/mL was utilized.

Results

During the titration of the monoclonal antibody 12C4 or 12C2, a positivereaction was indicated by the presence of a band on the top portion ofeach strip. The 2.1 mg/mL antibody concentration was selected, as itresulted in an intense signal with minimum background, and wasimmobilized at the test site of the membrane. The protocol consisted ofadding 20 μL serum (pre-diluted 1:10 in chase buffer or neat) at port202 immediately (<1 minute) followed by 150 μL chase buffer 218 at port217. The sample was left to incubate in device for 30 minutes at roomtemperature and ambient humidity.

The humanized anti-VLA-4 antibody, in sample matrix, IgG4 (myeloma),anti-VLA-4 together with IgG4, and the formulation buffer are tested ona flow system with 12C4 immobilized. The humanized anti-VLA-4 antibodywas tested at the concentration range of 0.25 and 100 μg/mL. The 12C4antibody was able to detect the humanized anti-VLA-4 antibody to aconcentration of 0.25 μg/mL.

The 12C4 antibody was then used to manufacture test cassettes. FIG. 2illustrates the specificity of the test utilizing the test cassettes.All tests showed a single band 215, indicating a valid test, but onlythe cassettes for the humanized anti-VLA-4 antibody test showed a secondband 214 indicating a positive test.

In some embodiments, the test includes additional components. Anti-VLA-1is a humanized monoclonal antibody to VLA-1. The constant region of someanti-VLA-1 antibodies is IgG1, while the variable antigen binding regionis of mouse origin. In some embodiments, anti-VLA-1 is used as anadditional control to assess specificity of the test system. Additionalcontrols that can be included in the test system include assessingtolerance of the test to excess levels of analytes that may be found inthe serum, e.g., IgG4, drug, or anti-drug antibodies. A reader can beused for semi-quantitative or quantitative measurement of anti-VLA4concentrations.

Example 3 Development of a Test to Assay Anti-VLA-4 Antibody UsingAnti-Anti-VLA-4 mAb-Gold Conjugates

Experiments were conducted to test the ability of a different format toeffectively detect anti-VLA-4 antibody in serum. The two part plasticcassette of this lateral flow test system is the same as that forExample 2 with a few alterations. Referring to FIG. 3, the top part 300contained three openings—one 302 is for the application of samples 310,opening 317—for application of chase buffer 318 and a window 303 forvisual detection of the results. The lower part 301 accommodated anitrocellulose test strip 304 on which the mouse 12C4 mAb 305 wasimmobilized at a “test site” (T) 306 and a goat anti-mouse antibody 307at a “control site” (C) 308.

Upstream from the test and control sites, there was a fibrous pad(“sample pad”) 309 that received the sample 310. Further upstream fromthe test and control sites there was a “gold pad” 311 of fibrousmaterial where a mouse anti-anti-VLA4 antibody 12C4 was conjugated togold particles, the complex being 312. The two-step assay was utilized.In the first step, sample 310 was applied to the sample opening 302 andmigrated toward the capture zone 305 ahead of the gold conjugate to forma complex of a sample 310 (anti-VLA4) with the mouse 12C4 or rat 12C2 ontest line 305. In the second step, the chase buffer 318 was applied tothe chase buffer port 317 and rehydrated the monoclonal mouseanti-anti-VLA4 antibody 12C4 gold conjugate 312. The conjugate 312 thentraveled toward the capture test line 305 to form a complex withanti-VLA-4 bound to capture line 305, and the reaction at site 306 wasvisualized as a red line 314.

Excess gold-labeled free antibody (mouse 12C4 mAb) traveled further tothe control site where a goat anti-mouse antibody 307 captured the mAb,giving rise to an additional visible red line 315. Any excess material,gold, and chase buffer was absorbed at the distal end of thenitrocellulose strip by an absorbent filter pad 316.

When using spiked natalizumab samples, this test format is morerestricted in its range of detection than the test conducted with theanti-human IgG4 gold conjugate because the complex at the test site onlyformed when the sample molecule can react with both the anti-(anti-VLA-4antibody)/gold conjugate and the anti-(anti-VLA-4 antibody) antibody atthe test site. In a simplistic representation, the anti-VLA-4 moleculeis bridging between the gold labeled anti-(anti-VLA-4 antibody) antibodyand the membrane immobilized anti-(anti-VLA-4 antibody) antibody.Therefore, this test is less sensitive for overall detection of theanti-VLA-4 antibody when using actual samples from patients. This formatresults in a more restricted test in that it detects only intactnatalizumab, which is an IgG4-based antibody. This format does notdetect half-antibody exchanged molecules, which is observed in patientstreated with natalizumab and other IgG4 antibodies. Therefore, the usesof this format are generally restricted to detection of intact drug.

Materials

Mouse anti-(anti-VLA-4 antibody) mAB 12C4 (Elan), 2.1 mg/mL

Humanized anti-VLA-4 Ab (Biogen Idec), 21 mg/mL

Humanized anti-VLA-4 Ab formulation buffer (Biogen Idec)

Goat anti-mouse antibody, 3.0 mg/ml

Chase buffer (PBS with 1% w/v BSA and 1% w/v Tween 20)

Gold conjugate (40 nm)

Test Development

A lateral flow immunochromatographic system was developed with the mAb12C4 directed against the humanized anti-VLA-4 antibody. To determinethe appropriate concentration of the capture antibody, 12C4 was titratedon separate test strips and tested against the humanized anti-VLA-4antibody at the concentrations ranging from 0.25 to 100 μg/mL. The 12C4antibody was then immobilized at the test site of the membrane at 2.1mg/mL. Also, 12C4 antibody was conjugated to 40 nm gold particles,lyophilized in the conjugate pad and utilized as a detector molecule. Atthe control site of the membrane, a goat anti-mouse antibody at 3.0mg/mL was utilized.

Results

During the titration of the monoclonal antibody 12C4, a positivereaction was indicated by the presence of a band on the top portion ofeach strip. The 2.1 mg/mL antibody concentration was selected, as itresulted in an intense signal with minimum background, and wasimmobilized at the test site of the membrane. The protocol consisted ofadding 20 μL serum (pre-diluted 1:10 in chase buffer or neat) at port302 immediately (<1 minute) followed by 150 μL chase buffer 318 at port317. The sample was left to incubate in device for 30 minutes at roomtemperature and ambient humidity.

The humanized anti-VLA-4 antibody in matrix, anti-VLA-4 antibody withadditional IgG4 added and the formulation buffer were tested on a flowsystem with 12C4 immobilized. The humanized anti-VLA-4 antibody wastested at the concentration range of 1 and 100 μg/mL. The 12C4 antibodywas able to detect the humanized anti-VLA-4 antibody to a concentrationof 1 μg/mL.

The 12C4 antibody was then used to manufacture test cassettes. FIG. 3illustrates the specificity of the test utilizing the test cassettes.All tests showed a single band 315, indicating a valid test, but onlythe cassette for the humanized anti-VLA-4 antibody test showed a secondband 314, indicating a positive test.

Example 4 Development of a Test to Assay Anti-Drug Antibody UsingAnti-VLA-4 Drug and Anti-VLA-4 Drug Gold Conjugate

Assays and methods to detect antibodies directed against a drug, e.g.,natalizumab are described herein. To test such an assay, the two-partplastic cassette of this lateral flow test system was the same as thatfor Examples 1-3 with a few alterations. Referring to FIG. 4, the toppart 400 contained three openings—opening 402 for the application ofsamples 410, opening 417—for chase buffer 418 and a window 403 forvisual detection of the results. The lower part 401 accommodated anitrocellulose test strip 404 on which the anti-VLA-4 antibody 405 wasimmobilized at a “test site” (T) 406 and a control antibody 407 at a“control site” (C) 408.

Upstream from the test and control sites, there was a fibrous pad(“sample pad”) 409 that received the sample 410. Further upstream fromthe test and control sites there was a “gold pad” 411 of fibrousmaterial where anti-VLA-4 was conjugated to gold particles, the complexbeing 412. The two-step assay was utilized. In the first step, sample410 was applied to the sample opening 402 and migrated toward thecapture zone 405 ahead of the gold conjugate to form a complex of asample 410 (anti-anti-VLA4) with the anti-VLA-4 on test line 405. In thesecond step, the chase buffer 418 was applied to the chase buffer port417 and rehydrated the anti-VLA-4 gold conjugate 412. The conjugate 412then traveled toward the capture test line 405 to form a complex withanti-(anti-VLA-4) bound to capture line 405, and the reaction at testsite 406 was visualized as a red line 414.

Any excess material, gold, and chase buffer was absorbed at the distalend of the nitrocellulose strip by an absorbent filter pad 416.

The specificity of the test was due to the fact that the complex at thetest site only formed when the sample molecule must bind to both theanti-VLA antibody on the test line and anti-VLA-4 gold conjugate. In asimplistic representation, the anti-(anti-VLA-4 antibody) antibody isbridging between the gold labeled anti-VLA-4 antibody and the membraneimmobilized anti-VLA-4 antibody, thus, in some cases, this type of assayis referred to as a “bridging assay”.

In some embodiments, excess gold-labeled and free anti-VLA-4 antibodycan travel further to the control site 408 where a control antibody 407is available to capture the matrix components, giving rise to anadditional visible red line 415.

A reader can be used for semi-quantitative determination of theconcentration of the tested analytes. To record the results of thelateral flow system, a reader based on visual positive/negative scoringwas used. The visual threshold on the reader corresponds to valueswithin the 20-40 unit range. If the signal was recorded visuallypositive within this range then it should be considered a very weak pinkline that may not be visible to all operators.

Materials

Mouse anti-(anti-VLA-4 antibody) mAB 12C4 (Elan), 2.1 mg/mL

Rabbit anti-(anti-VLA-4 antibody) mAB (Biogen Idec), 1.1 mg/mL

Rat anti-(anti-VLA-4 antibody) mAb 12C2 (Biogen Idec), 1.76 mg/mL

Humanized anti-VLA-4 Ab (Biogen Idec), 21 mg/mL

Humanized anti-VLA-4 Ab formulation buffer (Biogen Idec)

Control Antibody

Chase buffer (PBS with 1% w/v BSA and 1% w/v Tween 20)

Gold conjugate (40 nm)

Test Development

A lateral flow immunochromatographic system was developed with theanti-VLA-4 antibody directed against the anti-(anti-VLA-4 antibody)antibody. To determine the appropriate concentration of the captureantibody, anti-VLA-4 antibody was utilized, which was titrated onseparate test strips and tested against anti-(anti-VLA-4 antibody)antibody at a range of concentrations from 25 μg/mL to 0.5 μg/mL. Theanti-VLA-4 antibody was then immobilized at the test site of themembrane at 0.5 mg/mL. An anti-VLA-4 was conjugated to 40 nm goldparticles. The anti-VLA-4 immunogold conjugate was then lyophilized inthe conjugate pad and utilized as a detector molecule. At the controlsite of the membrane, control antibody was utilized.

Results

The 0.5 mg/mL antibody concentration was selected for immobilizationbased on titration experiments, as it resulted in an intense signal withminimum background. The protocol consisted of adding 20 μL serum(pre-diluted 1:10 in chase buffer or neat) at port 402 immediately (<1minute) followed by 150 μL chase buffer 418 at port 417. The sample wasleft to incubate in device for 30 minutes at room temperature andambient humidity.

The mouse and rabbit anti-(anti-VLA-4 antibody) antibody, humanizedanti-VLA-4 antibody, the combination of the above, and the formulationbuffer were tested on a flow system with anti-VLA-4 antibodyimmobilized. The anti-(anti-VLA-4 antibody) antibody was tested at theconcentration range of 25 μg/mL to 0.5 μg/mL. The humanized anti-VLA-4antibody was able to detect the human anti-(anti-VLA-4 antibody)antibody to a concentration of 0.5 μg/mL.

The humanized anti-VLA-4 antibody drug was then used to manufacture testcassettes. FIG. 4 illustrates the specificity of the test utilizing thetest cassettes. All tests showed a single band 415, indicating a validtest, but only the cassette for the anti-drug antibody test showed asecond band 414, indicating a positive test.

A control line or additional normalization line may be used for thepurpose of data normalization in the lateral flow assay. Thisnormalization line may be an immobilized antibody against one of thematrix (e.g., serum) components that have similar concentrations acrossindividual matrix samples. Alternatively, a normalization line may be animmobilized antibody against a protein from an irrelevant species thatis not present in the tested matrix, but added at the sameconcentrations to each matrix sample prior to placing it on a lateralflow device. This protein can be added to each sample directly, or, incase when samples are being diluted with a chase buffer, may be acomponent of the chase buffer. A reader can be used forsemi-quantitative or quantitative measurement of anti anti-VLA4concentrations.

Other Embodiments

It is to be understood that while the invention has been described inconjunction with the detailed description thereof, the foregoingdescription is intended to illustrate and not limit the scope of theinvention, which is defined by the scope of the appended claims. Otheraspects, advantages, and modifications are within the scope of thefollowing claims.

1-83. (canceled)
 84. A method of assaying the efficacy of a treatment,the method comprising: a) obtaining a biological sample from a subjectundergoing treatment to decrease the level of a humanized anti-VLA-4IgG4 antibody in the subject; b) contacting the sample with a captureagent associated with a substrate, wherein the capture agent can bind ahumanized anti-VLA-4 IgG4 antibody in the sample, and wherein thesubstrate is a portion of a lateral flow immunochromatographic systemcomprising, in order of flow, a label zone, a sample receiving zone, anda test zone configured such that: the label zone is located upstream ofthe sample receiving zone and contains one or more secondary agents, thetest zone is downstream of the label zone, upon flow through the lateralflow immunochromatographic system the sample reaches the test zonebefore a secondary agent from the label zone upon flow through thelateral flow immunochromatographic system, and upon flow of the samplethrough the system, humanized anti-VLA-4 IgG4 antibody from the samplewill first bind with a capture agent in the test zone to form a complex,the secondary agent will then reach the test zone, and then the complexwill bind with the secondary agent; and c) detecting binding of thehumanized anti-VLA-4 IgG4 antibody to the capture agent with a secondaryagent within the lateral flow immunochromatographic system anddetermining whether the level of the humanized anti-VLA-4 IgG4 antibodyis above or below a predetermined level based on the detecting, whereina level of the humanized anti-VLA-4 IgG4 antibody in the sample lessthan a predetermined level is indicative of the efficacy of thetreatment.
 85. The method of claim 84, comprising one, two, three, four,or all of the characteristics selected from the following: a) thepredetermined level is about 1 μg/ml, 2 μg/ml, 3 μg/ml, 4 μg/ml, 5μg/ml, 6 μg/ml, 7 μg/ml, 8 μg/ml, 9 μg/ml, 10 μg/ml, 15 μg/ml, or 20μg/ml, b) the biological sample is obtained from the subject one day,two days, three days, four days, five days, six days, one week, twoweeks, three weeks, or four weeks after the treatment, and/or c) thetreatment is repeated once a day, or once every two days, three days,four days, five days, six days, one week, two weeks, three weeks, orfour weeks, d) the biological sample is blood, plasma, serum, urine,saliva, cerebrospinal fluid, sputum, ocular lens fluid, sweat, milk,ascites fluid, mucous, synovial fluid, peritoneal fluid, transdermalexudates, pharyngeal exudates, bronchoalveolar lavage, trachealaspirations, semen, cervical mucus, vaginal secretion, urethralsecretion, or amniotic fluid, and e) the capture agent is an antibody oran antigen binding fragment thereof, or an integrin.
 86. The method ofclaim 84, wherein the treatment comprises blood apheresis selected fromis therapeutic plasma exchange, cytoreduction, photophoresis, andselective absorption.
 87. The method of claim 84, wherein the secondaryagent is: a) an anti-anti-VLA-4 antibody or antigen binding fragmentthereof, or b) an anti-IgG4 antibody or antigen binding fragmentthereof.
 88. The method of claim 84, wherein: a) the substrate isnitrocellulose, cellulose acetate, filter paper, cloth, or glass fiberpaper, and/or b) the binding of the capture agent to the antibody isdetectable within about 30 seconds to about 60 minutes or longer afterthe binding.
 89. The method of claim 84, wherein the subject was treatedwith natalizumab and has been diagnosed with or is suspected of having aJC virus infection.
 90. A kit for assaying the level of an antibody in abiological sample in a subject comprising: a) a tester comprising i) asubstrate; and ii) a detectable capture agent associated with thesubstrate wherein the capture agent can bind a humanized anti-VLA-4 IgG4antibody in the sample, and wherein the substrate is a portion of alateral flow immunochromatographic system comprising, in order of flow,a label zone, a sample receiving zone, and a test zone configured suchthat: the label zone is located upstream of the sample receiving zoneand contains one or more secondary agents, the test zone is downstreamof the label zone, upon flow through the lateral flowimmunochromatographic system the sample reaches the test zone before asecondary agent from the label zone upon flow through the lateral flowimmunochromatographic system, and upon flow of the sample through thesystem, humanized anti-VLA-4 IgG4 antibody from the sample will firstbind with a capture agent in the test zone to form a complex, thesecondary agent will then reach the test zone, and then the complex willbind with the secondary agent, and b) a chase buffer.
 91. The kit ofclaim 90, wherein the binding of the capture agent to the humanizedanti-VLA-4 IgG4 antibody produces a detectable signal.
 92. The kit ofclaim 90, wherein: a) the binding of the capture agent to the humanizedanti-VLA-4 IgG4 antibody is detected using the secondary agent b) thecapture agent is an antibody or antigen binding fragment thereof, or anintegrin, and/or c) the binding of the capture agent to the humanizedanti-VLA-4 IgG4 antibody is detectable within about 30 seconds to about60 minutes or longer after the binding.
 93. The kit of claim 90, whereinthe substrate is nitrocellulose, cellulose acetate, filter paper, cloth,or glass fiber paper.
 94. The kit of claim 90, wherein the biologicalsample is blood, plasma, serum, urine, saliva, cerebrospinal fluid,sputum, ocular lens fluid, sweat, milk, ascites fluid, mucous, synovialfluid, peritoneal fluid, transdermal exudates, pharyngeal exudates,bronchoalveolar lavage, tracheal aspirations, semen, cervical mucus,vaginal secretion, urethral secretion, or amniotic fluid.
 95. The kit ofclaim 90, wherein the secondary agent is: a) an anti-anti-VLA-4 antibodyor antigen binding fragment thereof, or b) an anti-IgG4 antibody orantigen binding fragment thereof.
 96. The kit of claim 95, furthercomprising instructions for one or more of the following: a)interpreting the level of the humanized anti-VLA-4 IgG4 antibody, b)interpreting the level of the anti-anti-VLA4 antibody or antigen bindingfragment thereof, c) interpreting the level of the anti-IgG4 antibody orantigen binding fragment thereof, and d) monitoring the efficacy oftreatment in the subject.
 97. A method of decreasing the level of ahumanized anti-VLA-4 IgG4 antibody in a subject, the method comprising:a) contacting a biological sample from a subject with a capture agentassociated with a substrate, wherein the capture agent can bind ahumanized anti-VLA-4 IgG4 antibody in the sample, and wherein thesubstrate is a portion of a lateral flow immunochromatographic systemcomprising, in order of flow, a label zone, a sample receiving zone, anda test zone configured such that: the label zone is located upstream ofthe sample receiving zone and contains one or more secondary agents, thetest zone is downstream of the label zone, upon flow through the lateralflow immunochromatographic system the sample reaches the test zonebefore a secondary agent from the label zone upon flow through thelateral flow immunochromatographic system, and upon flow of the samplethrough the system, humanized anti-VLA-4 IgG4 antibody from the samplewill first bind with a capture agent in the test zone to form a complex,the secondary agent will then reach the test zone, and then the complexwill bind with the secondary agent; b) detecting binding of thehumanized anti-VLA-4 IgG4 antibody to the capture agent with a secondaryagent within the lateral flow immunochromatographic system anddetermining whether the level of the humanized anti-VLA-4 IgG4 antibodyis above or below a predetermined level based on the detecting; and c)treating the subject until the level of the humanized anti-VLA-4 IgG4antibody in the sample reaches the predetermined level.
 98. The methodof claim 97, wherein the treatment comprises blood apheresis selectedfrom is therapeutic plasma exchange, cytoreduction, photophoresis, andselective absorption.
 99. The method of claim 97, wherein: a) thepredetermined level is about 1 μg/ml, 2 μg/ml, 3 μg/ml, 4 μg/ml, 5μg/ml, 6 μg/ml, 7 μg/ml, 8 μg/ml, 9 μg/ml, 10 μg/ml, 15 μg/ml, or 20μg/ml, b) contacting a biological sample from a subject with a captureagent associated with a substrate, detecting binding of humanizedanti-VLA-4 IgG4 antibody to the capture agent with a secondary agentwithin the lateral flow immunochromatographic system, and determiningwhether the level of the humanized anti-VLA-4 IgG4 antibody is above orbelow a predetermined level based on the detecting occurs one day, twodays, three days, four days, five days, six days, one week, two weeks,three weeks, or four weeks after a previous treatment, and/or c) thetreatment is repeated once a day, or once every two days, three days,four days, five days, six days, one week, two weeks, three weeks, orfour weeks.
 100. The method of claim 97, wherein the biological sampleis blood, plasma, serum, urine, saliva, cerebrospinal fluid, sputum,ocular lens fluid, sweat, milk, ascites fluid, mucous, synovial fluid,peritoneal fluid, transdermal exudates, pharyngeal exudates,bronchoalveolar lavage, tracheal aspirations, semen, cervical mucus,vaginal secretion, urethral secretion, or amniotic fluid.
 101. Themethod of claim 97, wherein: a) the capture agent is an antibody or anantigen binding fragment thereof, or an integrin, and/or b) the bindingof the capture agent to the antibody is detectable within about 30seconds to about 60 minutes or longer after the binding.
 102. The methodof claim 97, wherein the secondary agent is: a) an anti-anti-VLA-4antibody or antigen binding fragment thereof, or b) an anti-IgG4antibody or antigen binding fragment thereof.
 103. The method of claim97, wherein the substrate is nitrocellulose, cellulose acetate, filterpaper, cloth, or glass fiber paper.